Natural proteins differ from most polymers in that they predominantly populate a single, ordered three-dimensional structure in solution. It has long been recognized that this ordered structure can be transformed to an approximate random chain by changes in temperature, pressure or solvent conditions (Neurath et al., (1944) Chem. Rev. 34: 157–265). The ability to induce protein unfolding, and subsequent refolding, has allowed scientists to analyze the physical chemistry of the folding reaction in vitro (Schellman, (1987) Annu. Rev. Biophys. Bio. 16: 115–37). These investigations have shed light on the kinetics and thermodynamics of conformational changes in proteins and are of biological interest for two important reasons. First, they aid in the understanding of how proteins, which start their existence in the cell in a disordered state, manage to rapidly transform into a single, folded, functional conformation. Second, they elucidate the nature of functionally significant structural fluctuations present in proteins once folding equilibrium is reached.
The function of a protein is contingent on the stability of its native conformation. Consequently, in the field of protein biochemistry, stability measurements are frequently performed to establish a polypeptide as a stably folded protein and to study the physical forces that lead to its folding (Schellman, (1987) Annu. Rev. Biophys. Bio. 16: 115–37). Stability measurements also provide important biological information; a decrease in stability can be a sign of misfolding, which in some proteins leads to disease (Dobson, (1999) Trends Biochem. Sci. 24: 329–32) while an increase in stability can be indicative of ligand binding (Schellman, (1975) Biopolymers 14: 999–1018). Despite their utility, stability measurements currently necessitate time-consuming experiments with pure protein samples. In proteomic experiments (Blackstock & Weir, (1999) Trends Biotechnol. 17: 121–27), where a large number of polypeptides often need to be analyzed, stability measurements are not practical.
Recent studies have demonstrated that hydrogen exchange coupled with electrospray ionization (ESI) mass spectrometry can qualitatively distinguish native-like proteins from unfolded polypeptides in partially purified samples (Rosenbaum et al., (1999) J. Am. Chem. Soc. 121: 9509–13) and can be used to study the kinetics and thermodynamics of folding (Miranker et al., (1996) FASEB J. 10: 93–101; Deng & Smith, (1999) Anal. Biochem. 276: 150–60). However, these studies did not disclose the quantitative analysis of native-like proteins.
Stability measurements are frequently performed to establish a polypeptide as a stably folded protein and to study the forces that lead to its folding. Conventional denaturation methods used for the analysis of protein stability have at least the following identified experimental limitations: 1) stability can only be measured under conditions where the protein is partially unfolded; 2) the energetics of localized fluctuations can not be easily measured; 3) many proteins cannot be analyzed because they aggregate during the course of denaturation; 4) stability measurements cannot be obtained in complex mixtures, extracts or living cells; 5) relatively large amounts of protein are required for analysis; and 6) denaturation experiments are time-consuming and not amenable to high-throughput analysis. These constraints drastically limit the number of biological problems that can be addressed through stability measurements.
Thermodynamic stability is an important biological property that has evolved to an optimal level to fit the functional needs of proteins. Therefore, investigating the stability of proteins is important not only because it affords information about the physical chemistry of folding, but also because it can provide important biological insights. A proper understanding of protein stability is also useful for technological purposes. The ability to rationally make proteins of high stability, low aggregation or low degradation rates will be valuable for a number of applications. For example, proteins that can resist unfolding can be used in industrial processes that require enzyme catalysis at high temperatures (Van den Burg et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95(5): 2056–60); and the ability to produce proteins with low degradation rates within the cell can help to maximize production of recombinant proteins (Kwon et al., (1996) Protein Eng. 9(12): 1197–202).
Stability measurements can also be used as probes of other biolological phenomena. The most basic of these phenomena is biological activity. The ability of proteins to populate their native states is a universal requirement for function. Therefore, stability can be used as a convenient, first level assay for function. For example, libraries of polypeptide sequences can be tested for stability in order to select for sequences that fold into stable conformations and might potentially be active (Sandberg et al., (1995) Biochem. 34: 11970–78).
Changes in stability can also be used to detect binding. When a ligand binds to the native conformation of a protein, the global stability of a protein is increased (Schellman, (1975) Biopolymers 14: 999–1018; Pace & McGrath, (1980) J. Biol. Chem. 255: 3862–65; Pace & Grimsley, (1988) Biochem. 27: 3242–46). The binding constant can be measured by analyzing the extent of the stability increase. This strategy has been used to analyze the binding of ions and small molecules to a number of proteins (Pace & McGrath, (1980) J. Biol. Chem. 255: 3862–65; Pace & Grimsley, (1988) Biochem. 27: 3242–46; Schwartz, (1988) Biochem. 27: 8429–36; Brandts & Lin, (1990) Biochem. 29: 6927–40; Straume & Freire, (1992) Anal. Biochem. 203: 259–68; Graziano et al., (1996) Biochem. 35: 13386–92; Kanaya et al., (1996) J. Biol. Chem. 271: 32729–36).
The linkage between stability and binding has recently been implemented as a method to detect ligand binding (U.S. Pat. No. 5,679,582 to Bowie & Pakula). This method, however, does not take advantage of the high sensitivity available from an analytical technique such as MALDI mass spectrometry, and cannot be employed at the low protein levels that MALDI mass spectrometry can detect. Moreover, proteolytic methods can require additional steps to isolate and analyze proteolytic fragments and cannot be performed in an in vivo setting. Finally, this method cannot be employed to generate quantitative measurements of protein stability.
Thus, there remains substantial room for improvement in existing methods of screening for, quantitatively determining and beneficially employing protein stability measurements. A particularly desirable method would provide for the high throughput quantitative determination of protein stability, would require only very small quantities of sample, would permit in vivo measurement of protein stability and would generate data useful for a variety of stability-based applications. Until the disclosure of the present invention set forth herein, such a method was not available in the art.